Paper microfluidic devices for detection of improvised explosives

ABSTRACT

Paper microfluidic devices for testing for explosives are provided, along with methods of fabricating and using the same. One or more channels are formed on a paper substrate, and a test spot is formed in at least one of the channels. The channels can be hydrophobic. A test reagent is provided in the test spot and tests for explosives.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims the benefit of U.S. ProvisionalApplication Ser. No. 61/794,955, filed Mar. 15, 2013, which is herebyincorporated by reference herein in its entirety, including any figures,tables, and drawings.

GOVERNMENT SUPPORT

The subject invention was made with government support under a researchproject supported by National Institute of Justice Award No. NIJ20012-90426-FL-DN. The government has certain rights in this invention.

BACKGROUND

Improvised or homemade explosives (HMEs) were once limited to war zonesbut have recently become a concern for law enforcement and other firstresponders in the United States and abroad. Such responders mayencounter organized groups or curious “citizen scientists” synthesizingHMEs. Fast and accurate identification of the explosive compound used isof the utmost importance. Common constituents of HMEs include organicand inorganic compounds, sugars, and elemental metals. Many differentanalytical technologies exist for detecting and quantifying explosivematerials; however, the different unregulated and easily obtainedcompounds used in the devices vary greatly in molecular mass, structure,and physicochemical properties, and no single analytical instrument hasthe capability to identify them all.

A number of different techniques are available for the identification ofexplosive compounds. Gas chromatography/mass spectrometry (GC/MS),liquid chromatography/mass spectrometry (LC/MS), or Fourier transforminfrared spectroscopy (FT-IR) may be used for organic and inorganiccompounds while ion chromatography (IC) and capillary electrophoresis(CE) may be used for inorganic ions. Metals can be detected by scanningelectron microscopy with energy-dispersive X-ray spectroscopy (SEM/EDS)or by X-ray diffraction (XRD). Some compounds require electrosprayionization mass spectroscopy (ESI-MS) to be identified in a sample.These detection techniques have similar shortcomings; they requirelarge, expensive pieces of instrumentation that, with the exception ofFT-IR, are not portable. Due to vacuum, power, and gas requirements, theinstruments required for these techniques are necessarily centrallylocated. Thus, the sample must be collected and brought to thelaboratory, thereby increasing the amount of time before any analyticalinformation on the identity of the explosive can be obtained and, e.g.,provided to first responders.

Additionally, samples may need to undergo preparative techniques, suchas filtration or extraction before instrumental analysis can beperformed, thereby increasing the total analysis time. This alsoincreases the potential for analytes to be lost through such processesby adsorption onto the filtration medium or degradation of the sample byinteraction with the extraction solvent. Samples must also be preparedin sufficient volume (generally at least 200 μL per instrument) to behandled by an auto-sampler.

BRIEF SUMMARY

The subject invention provides devices and methods for detection ofexplosives, e.g., improvised explosives or homemade explosives (HMEs).In one embodiment, a paper microfluidic device (PMD) can be used todetect explosives, such as improvised explosives and/or HMEs.

In an embodiment, a PMD of the subject invention includes one or morehydrophobic channels on a paper substrate and a test reagent provided ata test spot of at least one of the hydrophobic channels. The testreagent is configured or adapted to test for one or more improvisedexplosives or HMEs. The test spot can be a colorimetric test spot.

In another embodiment, the subject invention provides a method oftesting a sample for explosives (e.g., improvised explosives and/orHMEs), wherein the method includes providing the sample to a PMD. ThePMD includes one or more hydrophobic channels on a paper substrate and atest reagent provided at a test spot of at least one of the hydrophobicchannels. The test reagent is configured or adapted to test forimprovised explosives or HMEs. The sample can be provided to the PMD ina very small amount (e.g., 50 μL or less). For example, the sample canbe provided to the PMD in a volume of 35 μL or about 35 μL.

In yet another embodiment, the subject invention provides a method offabricating a PMD, wherein the method includes printing a wax patternonto a paper substrate (e.g., filter paper or chromatography paper) andheating the paper substrate, thereby allowing the liquid wax topenetrate the paper substrate. One or more test reagents can be providedto test spots in the channels formed by the wax. The test reagent isconfigured or adapted to test for improvised explosives or HMEs.

In yet another embodiment, the subject invention provides a kit thatincludes a PMD. The PMD includes one or more hydrophobic channels on apaper substrate. The PMD may have test reagents already present for spottests or may have no test reagents present on the PMD. The kit mayadditionally include one or more test reagents. The test reagents areconfigured or adapted to test for improvised explosives or HMEs. A usercan apply the test reagents to the testing sites of a PMD that does notalready have the test reagents present.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show a process of fabricating a paper microfluidic device(PMD) according to an embodiment of the subject invention.

FIGS. 2A-2C show examples of the movement of samples through athree-dimensional PMD according to an embodiment of the subjectinvention.

FIG. 3 shows a PMD according to an embodiment of the subject invention.

FIG. 4 shows a PMD according to an embodiment of the subject invention.

FIG. 5 shows the results of a test of p-DMAC as a test reagent for ureanitrate.

FIG. 6 shows the results of a test of ammonium titanyl oxalate as a testreagent for peroxide.

FIG. 7 shows the results of a test for ammonium ion using differentsolvents.

FIG. 8A shows an image of a PMD according to an embodiment of thesubject invention.

FIG. 8B shows an image of a PMD according to an embodiment of thesubject invention.

FIG. 9 shows an image of a PMD according to an embodiment of the subjectinvention.

FIG. 10A shows an image of a PMD according to an embodiment of thesubject invention prior to analysis.

FIG. 10B shows an image of a PMD according to an embodiment of thesubject invention after analysis.

FIG. 11A shows an image of a PMD according to an embodiment of thesubject invention.

FIG. 11B shows an image of a PMD according to an embodiment of thesubject invention.

FIG. 11C shows an image of a PMD according to an embodiment of thesubject invention.

FIG. 11D shows an image of a PMD according to an embodiment of thesubject invention.

FIG. 11E shows an image of a PMD according to an embodiment of thesubject invention.

FIG. 12A shows an image of a PMD according to an embodiment of thesubject invention.

FIG. 12B shows an image of a PMD according to an embodiment of thesubject invention.

FIG. 12C shows an image of a PMD according to an embodiment of thesubject invention.

FIG. 13A shows an image of a PMD according to an embodiment of thesubject invention.

FIG. 13B shows an image of a PMD according to an embodiment of thesubject invention.

FIG. 14A shows an image of a PMD according to an embodiment of thesubject invention.

FIG. 14B shows an image of a PMD according to an embodiment of thesubject invention.

FIG. 14C shows an image of a PMD according to an embodiment of thesubject invention.

FIG. 14D shows an image of a PMD according to an embodiment of thesubject invention.

FIG. 14E shows an image of a PMD according to an embodiment of thesubject invention.

FIGS. 15A-15E show images of a PMD according to an embodiment of thesubject invention.

FIGS. 16A-16B show images of a PMD according to an embodiment of thesubject invention.

FIG. 17A shows an image of a PMD according to an embodiment of thesubject invention.

FIG. 17B shows an image of a PMD according to an embodiment of thesubject invention.

DETAILED DESCRIPTION

When the term “about” is used herein, in conjunction with a numericalvalue, it is understood that the value can be in a range of 95% of thevalue to 105% of the value, i.e. the value can be +/−5% of the statedvalue. For example, “about 1 kg” means from 0.95 kg to 1.05 kg.

Explosives, such as improvised explosives and homemade explosives(HMEs), can be made from organic compounds, inorganic compounds andions, sugars, and/or metals. Thus, these compounds are all important inexplosives analysis (e.g., improvised explosives analysis). However, nosingle existing instrument can detect all these groups. Instead, inexisting techniques, an unknown sample must be taken through a series ofextractions for analysis by different types of instrumentation.

Microfluidics involves the manipulation of fluids on a very small (e.g.,nanoliter) scale. Traditional microfluidic techniques use glass orpolydimethylsiloxane (PDMS) chips hooked to micro-scale pumps, valves,and power systems to transport a sample and effect separation beforereaching a detector such as a laser. By miniaturizing the system andexploiting the properties of fluids at the micro level, the samplevolume is reduced, the attendant reagent volume is similarly reduced,and the overall efficiency of the system is increased. PDMS chips mustbe painstakingly fabricated, resulting in a high attendant cost perchip, and they must be handled gently to avoid breakage. The need for apower system to drive the separation and detect the sample componentsmeans a traditional microfluidic system can only be used whereelectricity and a controlled environment are available.

Microfluidic principles have been applied to paper substrates to createpaper microfluidic devices. A wax pattern can be printed onto thesurface of filter paper, chromatography paper, or a nitrocellulosemembrane, and the paper (or membrane) can be heated so the liquid waxpenetrates the paper (or membrane), creating hydrophobic barriers thatfunction as lanes for the liquid sample to move along as it is routed tomultiplexed detection zones. An article by Martinez et al. presentsmicrofluidic paper-based analytical devices developed as healthcarediagnostic tools to be used in developing nations that do not possessthe infrastructure to maintain typical diagnostic equipment (Martinez etal., Diagnostics for the Developing World: Microfluidic Paper-BasedAnalytical Devices, Anal. Chem., 2010, 82, 3-10). The Martinez et al.article is hereby incorporated by reference in its entirety.

The following publications also discuss analysis techniques, explosives,or microfluidics: (Carrilho E et al., Understanding wax printing: Asimple micropatterning process for paper-based microfluidics, Anal.Chem. 2009, (81) 16; 7091-95); (Doctor, E.; McCord, B, Comparison ofAggregating Agents for Surface-Enhanced Raman Analysis ofBenzodiazepines, 64^(th) Annual meeting American Academy of ForensicSciences, Feb. 23, 2012); De Perre C et al., Rapid and specificdetection of urea nitrate and ammonium nitrate by electrosprayionization time-of-flight mass spectrometry using infusion with crownethers, Rapid Commun. Mass Spectrom. 2012; 26, 154-162); (Dungchai W etal., A low-cost, simple, and rapid fabrication method for paper-basedmicrofluidics using wax screen-printing. Analyst 2011; 136, 77-82);(Dungchai W et al. Use of multiple colorimetric indicators forpaper-based microfluidic devices, Analytica Chimica Acta 2010; 674,227-233); (Li X et al., Gold nanoparticle-based colorimetric assay forselective detection of aluminum cation on living cellular surfaces,Chem. Commun. 2010; 46, 988-990); Lu Y et al., Rapid prototyping ofpaper-based microfluidics with wax for low-cost, portable bioassay,Electrophoresis 2009; 30, 1497-1500); Martinez A W et al., Patternedpaper as a platform for inexpensive, low-volume portable bioassays,Angew. Chem. Int. Ed. 2007; 46, 1318-1320); (Bruce McCord; Inge Corbin;Edward Bender, Chromatography of Explosives In Forensic Investigationsof Explosives, 2^(nd) Edition, Beveridge, A. D., Ed. Taylor and Francis,Boca Raton Fla. 2012, pp. 585-620); (Greaux, J.; Lincoln, D.; McCord,BR., Separation and Detection of Smokeless Powder Additives byUltra-high Performance Liquid Chromatography with Tandem MassSpectrometry (UPLC/MS/MS), J. Forensic Sci. in press); (De Perre, C.,Prado, A. and McCord, B. R. Rapid and specific detection of urea nitrateand ammonium nitrate by electrospray ionization time-of-flight massspectrometry using infusion with crown ethers, Rapid Commun. MassSpectrom., 2011 26: 154-162); (Deperre, C.; McCord, B. Trace analysis ofurea nitrate by liquid chromatography-UV/fluorescence, Forensic ScienceInternational, 2011 211(1) 76-82); (Tarvin, M; McCord, B; Mount, K;Miller, M L, Analysis of Hydrogen Peroxide Field Samples by HPLC/FD andHPLC/ED in DC Mode Forensic Sci. Int. 2011 209(1-3):166-72); (Tarvin,M.; McCord, B.; Mount, K.; Sherlach K; Miller, M L, Optimization of TwoMethods for the Analysis of Hydrogen Peroxide: High Performance LiquidChromatography with Fluorescence Detection and High Performance LiquidChromatography with Electrochemical Detection in Direct Current Mode, J.Chromatogr. A 2010 1217(48) 7564-72); (Megan Bottegal; Gui-hua LisaLang; Mark Miller; Bruce McCord, Analysis of Ascorbic Acid-Based BlackPowder Substitutes by High Performance LiquidChromatography-Electrospray Ionization-Quadrupole Time-of-Flight MassSpectrometry (HPLC-ESI-QToFMS), Rapid Communications in MassSpectrometry 2010 24(9) 1377-1386); (Kristy G. Lahoda; Olivier L.Collin; John A. Mathis; Holly E. LeClair; Steven H. Wise and Bruce R.McCord, A Survey of Background Levels of Explosives and RelatedCompounds in the Environment, J. Forensic Sciences, 2008 53(4) 802-806);(Collin, O. L., Niegel, C. DeRhodes, K., McCord, B., Jackson, G., FastGC of Explosive Compounds using a Pulsed Discharge Electron CaptureDetector, J Forensic Sciences, 2006, 51 (4), 815-818); (Hopper, K.;McCord, B. “A Novel Method for Simultaneous Analysis of Cations andAnions in Inorganic Explosive Residue,” Talanta, 2005, 67, 304-312);(Mathis, J.; McCord, B. The Analysis of High Explosives by LiquidChromatography Electrospray Ionization Mass Spectrometry: MultiplexedDetection of Negative Ion Adducts, Rapid Communications in MassSpectrometry 2005, 19(2), 99-104); (Mathis, J.; McCord, B. A GradientReversed Phase High Performance Liquid Chromatography-ElectrosprayIonization Mass Spectrometric Method for the Comparison of SmokelessPowders Journal of Chromatography A, 2003, 988, 107-116); (Heramb, R.;McCord, B. Smokeless powders and their analysis, a brief review.Forensic Science Communications 2002, 4 (2), 1-7); (Wissinger, C. E.;McCord, B. R. A reversed phase HPLC procedure for smokeless powdercomparison, Journal of Forensic Sciences 2002, 47 (1), 168-174); (Doyle,J. M.; Miller, M. L.; McCord, B. R.; McCollam, D. A.; Mushrush, G. W. AMulticomponent Mobile Phase for Ion Chromatography Applied to theSeparation of Anions from the Residue of Low Explosives, AnalyticalChemistry 2000, 72(10), 2303-2307); (Smith, K. D.; McCord, B. R.;MacCrehan, W. A.; Mount, K.; Rowe, W. F. Detection of Smokeless PowderResidue on Pipe Bombs by Micellar Electrokinetic Chromatography, Journalof Forensic Sciences 1999, 44(4), 789-794); Doyle, J. M.; McCord, B. R.Novel electrolyte for the analysis of cations in low explosive residueby capillary electrophoresis Journal of Chromatography B 1998, 714,105-111); (McCord, B. R.; Hargadon, K.; Hall, K.; Burmeister, S.Forensic Analysis of Explosives using Ion Chromatographic Methods,Analytica Chimica Acta 1994, 288, 43-56); and (McCord, B. R.;Whitehurst, F. W. The Analysis and Characterization of TNT using LiquidChromatography with Photodiode Array Detection, Journal of ForensicSciences 1992, 37(6), 1574-1584). Each of these publications is herebyincorporated by reference in their entireties.

The subject invention is drawn to devices and methods for detection ofexplosives, e.g., improvised explosives or HMEs. In an embodiment of thesubject invention, a paper microfluidic device (PMD) is used to detectexplosives, such as improvised explosives and/or HMEs. The PMD can beused as an immediate-response field screening tool for explosives (e.g.,improvised explosives and/or HMEs) and their constituent ions.Advantageously, a very small extraction volume can be used, such as 50μL or less, for a sample. The sample can be used by personnel in thefield for immediate information or by analysts in the laboratory totriage samples and prioritize analyses. In one embodiment, an extractionvolume of 35 μL or about 35 μL is used as a sample.

According to several embodiments of the subject invention, papermicrofluidics is used for detection of improvised explosives or HMEs.Paper microfluidics is a transformational technology that permits thedevelopment of very inexpensive analytical devices. In an embodiment ofthe subject invention, a design is printed in ink (e.g., wax-based ink)on paper (e.g., filter paper or chromatography paper). The channels(e.g., wax channels) in the paper direct a liquid sample toward one ormore individual sections of the paper, which can contain at least onetest reagent. The channels can be hydrophobic. In certain embodiments,one or more of the test reagents can be a colorimetric test reagent. Ina particular embodiment, a nitrocellulose membrane can be used insteadof paper.

Colorimetric spot tests are a fundamental technique that is known invarious forms. Using physical and chemical properties of a chosenanalyte, spot tests react to give a visible color change when theanalyte of interest is present. Color spot tests may be presumptive orspecific, and can be used according to the subject invention in theanalysis of explosives. They may test for, e.g., intact compounds, ions,functional groups, metals, and/or enzymes. They may be based inclassical wet chemistry methods, nanoparticle aggregation, orantibody/aptamer detection. Color test kits for the analysis ofnitroaromatics, nitrate esters, and nitramines using spray reagents canbe used. Spot tests for compounds such as urea nitrate and hydrogenperoxide can be used in embodiments of the subject invention.

Colorimetric bioassay techniques can also be employed for smallmolecules and ions. For example, perchlorate can be detected bycolorimetric bioassay using perchlorate reductase enzyme and N-methylphenazinium sulfate as a visualizing agent. A nitrate reduction test canbe used to show whether bacteria can produce nitrate reductase enzyme toreduce nitrate to nitrite using sulfanilic acid andN,N-dimethyl-1-naphthalene. This technique, or a reverse thereof usingnitrate reductase, can be used.

Functionalized gold nanoparticles are another avenue of detection thatcan be used in embodiments of the subject invention. While most commonlyused in biomedical applications and DNA analysis, by adding a suitablereagent during the synthesis process, it is possible to createfunctionalized nanoparticles to detect a variety of compounds by theaggregation or disaggregation of gold nanoparticles that results in acharacteristic red-to-blue color change. Functionalized goldnanoparticles can be used for the detection of, among other things,aluminum, hydrogen peroxide, and reducing sugars including sucrose(table sugar).

By using multiple indicators for the same compound, a user's ability todiscriminate between different shades and intensities of color thatindicate concentration is increased, and data can be more accuratelyinterpreted. In the case of ions and small molecules, multipleindicators for the same compound can also help avoid a false positiveresult since the interferences for tests using different reagents ormechanisms for the same analyte are unlikely to be the same for bothsystems. Referring to FIG. 3, a PMD can include multiple redundant testson a single device. Change in the chemistry can permit detection of thesame compound with differing responses. The example in FIG. 3 showssimultaneous detection of two different compounds using five channels,two for one compound and three for the other. In FIG. 3, W representsthe width of the PMD, and L represents the length (or height).

The area of a PMD according to embodiments of the subject invention canbe less than 4.5 square centimeters (cm²). The area of a PMD accordingto embodiments of the subject invention can be less than, for example,50 cm², 20 cm², 10 cm², 7 cm², 6.5 cm², 6 cm², 5 cm², 4.5 cm², 4.25 cm²,4.2 cm², 4.1 cm², 4 cm², 3.5 cm², 3 cm², 2 cm², 1 cm², or 0.5 cm². A PMDaccording to embodiments of the subject invention can have a widthand/or length (height) of 2 centimeters (cm), 2.5 cm, 2.54 cm, or about2 cm. A PMD according to embodiments of the subject invention can have awidth and/or length (height) of, for example, 0.5 cm, 1 cm, 1.5 cm, 2cm, 2.5 cm, 2.54 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, or 10 cm.In one embodiment, a PMD has a width of about 2 cm and a length of about2 cm, giving an area of about 4 cm².

In certain embodiments, a single PMD can perform multiple analyses byhaving multiple channels and multiple test reagents. For example, apaper microfluidic device can perform, two, three, four, five, six,seven, eight, nine, 10, or more analyses on one device. These analysescan be performed simultaneously or essentially simultaneously (i.e.,after providing a single sample to a PMD and allowing it to travelthrough channels to multiple testing points). Such a PMD can be made ofpaper (e.g., chromatography paper) and wax, and can include smallquantities of test reagents. Each of these components is inexpensive;thus, a PMD according to embodiments of the subject invention canperform multiple simultaneous analyses (e.g., 2, 5, 10, or more) whilecosting only pennies. A PMD of the subject invention is also easilystored for long-term performance. Due to these factors, PMDs of thesubject invention are very advantageous for on-site forensic testing andscreening for improvised explosives and HMEs.

In an embodiment, a PMD can have a path that branches out into two ormore paths. Each branched path can lead to a testing spot, which caninclude a test reagent. Referring again to FIG. 3, a PMD having suchbranched paths is shown. Though a PMD with five branches is shown, thisshould not be construed is limiting. For example, PMDs having two,three, four, six, etc. branches can be used.

PMDs of the subject invention provide police and forensic evidencecollection teams an easily-stored and reliable tool for presumptivetesting of unknown evidence. The devices can be easily and cheaplymanufactured and take up no more space than a package of cigarettepapers. The low cost and easy portability allow for an analyst or crimescene response team member to carry a kit capable of instantidentification of unknown or hazardous compounds. PMDs of the subjectinvention can also be used in the analysis of seized drugs from, forexample, clandestine manufacturing sites or in a Customs setting in aport.

In several embodiments, a PMD is configured for the presumptivedetection of explosives (e.g., improvised explosives or HMEs). Thedevice can be configured to analyze samples from both pre- andpost-blast residues.

In an embodiment, a PMD includes colorimetric spots for forensicexplosives analysis. Colorimetric test reagents are present on thedevice, and such colorimetric test reagents can be present at or nearthe ends of channels formed in the device. Examples of colorimetric testreagents that can be included on a device include Greiss reagents,diphenylamine reagents, p-dimethylaminocinnamaldehyde (p-DMAC), andammonium titanyl oxalate. For example, Greiss and diphenylamine reagentscan be used to test for organic and inorganic nitrates, p-DMAC can beused to test for urea nitrate, and ammonium titanyl oxalate can be usedto test for peroxides. These reagents are given for exemplary purposesonly and should not be construed as limiting.

In certain embodiments, test reagents are spotted in an analysis lane ofa PMD. When the solvent containing the sample moves through the printedlane, it dissolves the reagents, allowing them to react with the sampleand generate a color change. The PMD can combine a set of tests formultiple compounds, including for example, chlorates, nitrates, organicnitrates, peroxides, fertilizer-based explosives, and/or highexplosives. Such a set of tests can be combined on a single piece ofpaper. The device can be configured for long-term storage with sensitiveand reliable detection of the relevant materials.

In several embodiments, a PMD is configured to provide fast andsensitive colorimetric detection of the constituents of improvisedexplosives or HMEs in pre- or post-blast settings. The PMD can be usedas a first-line response and presumptive field identification tool forthe presence of improvised explosives or HMEs before samples are sent toa central laboratory for analysis. In a laboratory setting, analysts canuse a PMD of the subject invention to triage evidence and prioritizeinstrumental analyses to maximize the amount of information generatedfrom a limited sample volume.

A PMD of the subject invention can be fabricated by, for example, plasmaetching, photolithography, PDMS printing, or wax printing. In apreferred embodiment, a PMD is fabricated by wax printing. A PMD made byphotolithography can be more finely resolved than one made by a waxprinting process, but the wax printing process does not require thespecialized equipment and personnel trained in the maintenance and useof that equipment that the other processes do.

In an embodiment, a PMD is fabricated by printing a wax pattern onto apaper substrate (e.g., filter paper or chromatography paper), and thepaper substrate can be heated so the liquid wax penetrates the papersubstrate. This creates hydrophobic barriers that function as lanes orchannels for a liquid sample to move along as it is routed to one ormore detection zones. This process is depicted in FIGS. 1A-1D. Thedesign can be conceptualized (FIG. 1a ), and the wax can be printed ontothe paper substrate (FIG. 1b ). The paper substrate can then be heatedso the liquid wax penetrates the paper substrate and creates hydrophobicbarriers (FIG. 1c ). Then, a liquid sample can move along the resultingas lanes or channels to one or more detection zones (FIG. 1d ).

The natural wicking action of the paper as it draws up the liquid sampletakes the place of a pump, and test reagents placed precisely within thepattern give an immediate, visible color change showing the presence ofan analyte.

In an embodiment, a PMD can have a three-dimensional (3-D)configuration. By using a device with a 3-D configuration, the number oftests incorporated can be increased while keeping reagent volume to aminimum. This can be accomplished using a chip, which can be produced ona single sheet of flat paper, which is then folded in a prescribed wayto line up the channels and reservoirs in the different layers. Thesample solution can be introduced into the reservoirs on the top layerof the device. The sample solution can be carried through the layers ofthe device by capillary action to interact with the test reagents. In aparticular embodiment, the folded PMD is inserted into a metal frame toensure the channels and reservoirs do not shift during use.

Referring to FIGS. 2A-2C, an example of the movement of samples througha 3-D PMD is depicted. When the analysis is complete, the PMD can beremoved from the frame (if a frame was used) and unfolded to view theresults. For clarity, the PMD can be marked with indicators to identifyeach test. FIG. 2a shows an example of a patterned paper. FIG. 2b showsa folded 3-D PMD which can then be injected with a test solution(sample). FIG. 2c shows an example of results of a test, where colorsindicate whether a target analyte is present. Though the boxes in FIGS.2a and 2c may appear to be different sizes and/or shapes, in a 3-D PMDthey are actually all approximately the same size and shape. In FIG. 2b, W represents the width of the 3-D PMD, L represents the length of the3-D PMD, and T represents the thickness of the 3-D PMD.

The volume of a 3-D PMD according to embodiments of the subjectinvention can be less than 4 cubic centimeters (cc). The volume of a 3-DPMD according to embodiments of the subject invention can be less than,for example, 20 cc, 15 cc, 10 cc, 7 cc, 5 cc, 4 cc, 3.5 cc, 3.2 cc, 3cc, 2 cc, 1 cc, 0.5 cc, or 0.2 cc. A 3-D PMD according to embodiments ofthe subject invention can have a width and/or length of 2.5 cm, 2.54 cm,or about 2.5 cm. A 3-D PMD according to embodiments of the subjectinvention can have a width and/or length of, for example, 0.5 cm, 1 cm,1.5 cm, 2 cm, 2.5 cm, 2.54 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm,or 10 cm. A 3-D PMD according to embodiments of the subject inventioncan have a thickness of 0.5 cm or about 0.5 cm. A 3-D PMD according toembodiments of the subject invention can have a thickness of, forexample, 0.1 cm, 0.5 cm, 1 cm, 1.5 cm, 2 cm, 2.5 cm, 2.54 cm, 3 cm, 4cm, or 5 cm. In one embodiment, a 3-D PMD has a width of about 2.5 cm, alength of about 2.5 cm, and a thickness of about 0.5 cm, giving a volumeof about 3.125 cc.

In one embodiment, the volume of a sample provided to a 3-D PMD is about75 μL. In an embodiment, a kit can include a PMD as described herein.The PMD may have test reagents already present for spot tests or mayhave no test reagents present on the PMD. The kit may additionallyinclude one or more test reagents for testing for compounds ofexplosives (e.g., improvised explosives and HMEs). A user can apply thetest reagents to the testing sites of a PMD that does not already havethe test reagents present.

In an embodiment, a method of testing a sample for explosives (e.g.,improvised explosives and/or HMEs) can include providing the sample to aPMD as described herein. The sample can be, for example, dissolved in asuitable solvent and applied to the PMD, though embodiments are notlimited thereto. The sample can be provided to the PMD in a very smallamount. In various embodiments, the amount of the sample provided to thePMD can be, for example, any of the following values, about any of thefollowing values, less than any of the following values, or at least anyof the following values (all numerical values are in μL): 0.001, 0.01,0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 70, 75, 80, 90, 100, 110, 120, 130, 140, 150, or 200. Forexample, the sample can be provided to the PMD in an amount of 75 about75 μL, less than 75 μL, 50 μL, about 50 μL, less than 50 μL, 35 μL,about 35 μL, or less than 35 μL. In some embodiments, the amount ofsolvent (having the sample dissolved therein) required to transport thesample to the test spot(s) can be 50-200 μL.

Examples of solvents that can be used include, but are not limited to,water, crown ethers, acetone, ethanol, methanol, dimethyl sulfoxide(DMSO), dimethylformaldehyde (DMF), and any combination thereof.

In an embodiment, a sample can be tested using a PMD as describedherein, and the results can be photographed. The image or images can betransmitted to a remote location, e.g., to a laboratory having an expertwho evaluates the results and responds with a course of treatment if itis required. This way, an expert need not be on site to interpret theresults of the tests. Such a picture can be taken with any suitabledevice, such as a camera of a mobile phone.

In a further embodiment, the image or images can be accompanied by oneor more images of standards run in the field, or such standard runimages can be provided before or after the image or images of the PMDtest.

Embodiments of the subject invention can advantageously allow for thecolorimetric detection of one or more analytes simultaneously oressentially simultaneously. Certain embodiments can also have redundanttesting for one or more analytes simultaneously or essentiallysimultaneously. This technology is very advantageous for firstresponders and forensic professionals who may encounter improvisedexplosives and HMEs in routine work. PMDs of the subject invention candetect a wide variety of compounds with very dissimilar properties. Theuse of PMDs in the field with all their attendant benefits—speed, lowcost, portability, specificity, sensitivity, ease of use, and ability togenerate results without electricity or specialized equipment—is limitedonly by the choice of compounds one wishes to detect. PMDs of thesubject invention are also advantageous for smaller labs that lack thenecessary funds or caseload to justify the dedicated equipment (e.g.,ion chromatography and specialized negative ion GC/MS systems andcapillaries) necessary for explosives analysis.

Embodiments of the subject invention provide police and forensicevidence collection teams easily-stored and reliable tools forpresumptive testing of unknown evidence. PMDs of the subject inventioncan be easily and cheaply manufactured and are very small and portable(e.g., a group may be about the size of a package of cigarette papers,or smaller). The low cost and easy portability allows analysts and crimescene response team members to carry a kit capable of instantidentification of unknown or hazardous compounds. PMDs of the subjectinvention can also be applied in the determination of seized drugs from,for example, clandestine manufacturing sites or a Customs setting in aport.

FIGS. 8, 9, and 17 show images of PMDs according to embodiments of thesubject invention. Referring to FIG. 8A, in an embodiment, a PMD canhave a single channel or lane. Referring to FIG. 8B, in an embodiment, aPMD can have five channels. Referring to FIG. 9, a PMD can be insertedinto a solution (e.g., in a vial) including the sample dissolved in asolvent. Referring to FIG. 17A, in an embodiment, a five-channel PMD canbe configured for detection of inorganic explosives detection with atotal run time (for the testing) of about 18 minutes. Referring to FIG.17B, in an embodiment, a five-channel PMD can be configured fordetection of inorganic explosives detection with a total run time (forthe testing) of about five minutes and can have a much smaller size thanthe PMD shown in FIG. 17A.

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allsolvent mixture proportions are by volume unless otherwise noted.

Example 1

A design in wax ink was printed on an 8.5-inch×11-inch sheet ofchromatography paper. To create the hydrophobic channels in the paper,the wax was heated and reflowed into the paper by running the printedsheet through a standard office document laminator. The finished chipswere then tested using a water solution of eosin Y indicator to ensurethe wax had penetrated the paper completely. A picture of this PMD isshown in FIG. 4. Referring to FIG. 4, this particular PMD has indicatingdye and can perform five simultaneous tests. The combination of heat andpressure the laminator applies to the printed chip creates hydrophobicsample lanes.

Several colorimetric tests were evaluated for their fitness for use inPMDs. Some reagents, such as p-DMAC for the detection of urea nitrate,are amenable to dissolution in a small amount of solvent and directspotting on the PMD. Likewise, peroxide can be detected by the use ofammonium titanyl oxalate on a paper substrate. Referring to FIG. 5, ureanitrate was detected with p-DMAC, giving a red color. Referring to FIG.6, peroxide was detected with ammonium titanyl oxalate, giving a yellowcolor.

Other tests were also evaluated on a paper substrate. A test forammonium ion gave a positive result when the ammonium source wasdissolved in various solvents. Referring to FIG. 7, ammonium ion wasdetected in deionized water (far left), acetone (second from left), anda mixture of 95% acetone and 5% methyl cellosolve (far right). Anegative result was obtained using methanol as a solvent.

Test reagents were spotted in analysis lane of the PMD. Thus, if asolvent containing a sample were moved through the printed lane, itwould dissolve the reagents, allowing them to react with the sample andgenerate a color change. The PMD combined a set of tests for ureanitrate and peroxide.

Example 2

Referring to FIGS. 10A and 10B, a five-channel PMD was configured totest for NH₄ ⁺, NO₃ ⁻, NO₂ ⁻, ClO₃ ⁻, and ClO₄ ⁻. FIG. 10B shows the PMDof FIG. 10A after a testing time of 18 minutes. Color change startedafter about five minutes.

Example 3

Five single-channel PMDs were tested. All of the PMDs were preparedusing 2 μL of each reagent spotted onto the PMD. Explosive compoundswere inserted in a plastic vial with 1000 ppm of the compound dissolvedin deionized water and deionized water was used as a blank. The reactiontime for the single lane PMDs was about five minutes.

FIGS. 11A-11B show the results the left side of each figure shows thechannel after testing the blank, and the right side of each figure showsthe channel testing the explosive compound. Referring to FIG. 11A, a PMDwas tested for chlorate (ClO₃ ⁻) using aniline sulfate and 50% sulfuricacid as reagents. The test spot changed from colorless to dark greenwhen the explosive compound was tested. Referring to FIG. 11B, a PMD wastested for nitrite (NO₂ ⁻) using the Greiss test with sulphanilic acidand 1-napthylamine as reagents. The test spot changed from colorless toorange/brown when the explosive compound was tested. Referring to FIG.11C, a PMD was tested for ammonium (NH₄ ⁺) using Nessler's Reagent as areagent. The test spot changed from pale yellow to orange/brown when theexplosive compound was tested.

Referring to FIG. 11D, a PMD was tested for nitrate (NO₃ ⁻) using (1) apaste of saturated tehalose solution with solid reducing mixture ofsulfanilic acid, sodium acetate, and zinc powder, (2) 20% sulfuric acid,and (3) 1-napthol as reagents. The test spot changed from colorless toorange when the explosive compound was tested. Referring to FIG. 11E, aPMD was tested for perchlorate (ClO₄ ⁻) using 0.05% methylene blue as areagent. The test spot changed from pale blue to purple when theexplosive compound was tested.

Example 4

Referring to FIGS. 12A-12C, five-channel PMDs were configured to testfor NO₃ ⁻ and NO₂ ⁻. FIG. 12A shows a PMD after the test run with 1000ppm potassium nitrite in deionized water. The nitrate test generated adark purple color while the nitrite test produced an orange-brown color.FIG. 12B shows a PMD after the test run with 1000 ppm potassium nitratein deionized water. The nitrate test generated an orange color while thenitrite test did not show any color change. FIG. 12C shows a PMD afterthe test run with 1000 ppm mixture of potassium nitrate and potassiumnitrite. The nitrite test produced an orange-brown color while thenitrate test showed a dark purple and orange color.

Example 5

Referring to FIGS. 13A and 13B, a five-channel PMD was configured totest for RDX, TNT, UN, NO₃ ⁻, and H₂O₂. FIG. 13B shows the PMD of FIG.13A after the test. A mixture of HMX, TNT, urea nitrate, and hydrogenperoxide in 50% acetone/50% water was tested. Color changes began tooccur after five minutes with a total run time of approximately 18minutes for complete visualization.

Example 6

Five single-channel PMDs were tested. All of the PMDs were preparedusing 2 μL of each reagent spotted onto the PMD. Explosive compoundswere inserted in a plastic vial with 1000 ppm of the compound dissolvedin 50% acetone/50% water and 50% acetone/50% water was used as a blank.The reaction time for the single lane PMDs was about five minutes.

FIGS. 14A-14B show the results; the left side of each figure shows thechannel after testing the blank, and the right side of each figure showsthe channel testing the explosive compound. Referring to FIG. 14A, a PMDwas tested for nitrate (NO₃ ⁻) using (1) a paste of saturated tehalosesolution with solid reducing mixture of sulfanilic acid, sodium acetate,and zinc powder, (2) 20% sulfuric acid, and (3) 1-napthol as reagents.The test spot changed from colorless to orange when the explosivecompound was tested. Referring to FIG. 14B, a PMD was tested fortrinitrotoluene (TNT) using 1.5 M potassium hydroxide as a reagent. Thetest spot changed from colorless to pink/red when the explosive compoundwas tested. Referring to FIG. 14C, a PMD was tested for hydrogenperoxide (H₂O₂) using ammonium titanyl oxalate as a reagent. The testspot changed from colorless to yellow when the explosive compound wastested. Referring to FIG. 14D, a PMD was tested for RDX, HMX, and PETNusing (1) zinc powder in 50% acetic acid, (2) sulfanilic acid, and (3)1-napthylamine as reagents. The test spot changed from colorless to pinkwhen the explosive compound was tested. Referring to FIG. 14E, a PMD wastested for urea nitrate (UN) using para-dimethylaminocinnamaldehyde as areagent. The test spot changed from yellow to red when the explosivecompound was tested.

Example 7

Referring to FIGS. 15A-15C, single-channel PMDs were configured to testfor UN. FIG. 15A shows the results of a blank run with just 50%acetone/50% water. FIG. 15B shows the results of a test run with 1000ppm UN in 50% acetone/50% water producing a red color. FIG. 15C showsthe results of a test run with 1000 ppm urea in 50% acetone/50%deionized water.

Example 8

Referring to FIGS. 16A and 16B, single-channel PMDs were configured totest for RDX/HMX/PETN. FIG. 16A shows the results of a test run with1000 ppm HMX in 50% acetone/50% water producing a pink color change.FIG. 15B shows the results of a test run with 1000 ppm potassium nitratein 50% acetone/50% water producing a noticeably different orange colorchange.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

What is claimed is:
 1. A paper microfluidic device (PMD), comprising: apaper substrate; one or more hydrophobic channels on the papersubstrate; and a test reagent provided at a test spot of at least one ofthe hydrophobic channels, wherein the test reagent is configured to testfor explosives.
 2. The PMD according to claim 1, comprising at least twohydrophobic channels on the paper substrate, wherein each hydrophobicchannel comprises a test spot, and wherein each test spot comprises atest reagent configured to test for at least one of improvisedexplosives and homemade explosives.
 3. The PMD according to claim 2,wherein at least two of the test spots have the same test reagentprovided thereon.
 4. The PMD according to claim 2, wherein a firsthydrophobic channel of the at least two hydrophobic channels comprises afirst test spot having a first test reagent configured to test for atleast one of improvised explosives and homemade explosives, wherein asecond hydrophobic channel of the at least two hydrophobic channelscomprises a second test spot having a second test reagent configured totest for at least one of improvised explosives and homemade explosives,and wherein the first test reagent is different from the second testreagent.
 5. The PMD according to claim 4, wherein a third hydrophobicchannel of the at least two hydrophobic channels comprises a third testspot having a third test reagent configured to test for at least one ofimprovised explosives and homemade explosives, and wherein the thirdtest reagent is different from the first test reagent and the secondtest reagent.
 6. The PMD according to claim 1, comprising a mainhydrophobic channel and a plurality of secondary hydrophobic channelsbranching off from the main hydrophobic channel, wherein each secondaryhydrophobic channel comprises a test spot having a test reagentconfigured to test for at least one of improvised explosives andhomemade explosives.
 7. The PMD according to claim 1, wherein eachhydrophobic channel is a wax channel, and wherein the paper substrate isfilter paper or chromatography paper.
 8. The PMD according to claim 1,wherein the area of the paper substrate having the one or morehydrophobic channels is less than 4.5 square centimeters (cm²).
 9. ThePMD according to claim 1, wherein the test reagent is a colorimetrictest reagent.
 10. The PMD according to claim 1, wherein the test reagentcomprises at least one of the following: functionalized goldnanoparticles; a mixture of perchlorate reductase and N-methylphenazinium sulfate; or a mixture of sulfanilic acid andN,N-dimethyl-1-naphthalene.
 11. A method of testing a sample forexplosives, comprising: providing the sample to the PMD according toclaim
 1. 12. The method according to claim 11, further comprisingdissolving the sample in a solvent to give a sample solution, whereinproviding the sample to the PMD comprises providing the sample solutionto the PMD in a volume of less than 75 pt.
 13. The method according toclaim 12, further comprising: photographing the PMD after the samplesolution has interacted with the test reagent to give a result image;and transmitting the result image to a remote location.
 14. Athree-dimensional (3-D) paper microfluidic device (PMD), comprising: apaper substrate folded into a 3-D configuration to give a plurality oflayers; a plurality of channels on the paper substrate; and a pluralityof reservoirs on the paper substrate, wherein the paper substrate isfolded such that each reservoir lines up with a channel on a differentlayer of the paper substrate than the reservoir is on, and wherein atleast one reservoir comprises a test reagent configured to test forexplosives.
 15. The 3-D PMD according to claim 14, wherein each channelis hydrophobic and each reservoir is hydrophobic, and wherein the testreagent is configured to test for at least one of improvised explosivesand homemade explosives.
 16. The 3-D PMD according to claim 14, whereinthe volume of the 3-D PMD is less than 4 cubic centimeters (cc).
 17. The3-D PMD according to claim 14, comprising at least three layers, whereina top layer of the 3-D PMD comprises at least one reservoir, wherein abottom layer of the 3-D PMD comprises at least one reservoir lined upwith the at least one reservoir of the top layer, wherein each layerbetween the top layer and the bottom layer comprises a channel lined upwith the at least one reservoir of the top layer and the at least onereservoir of the bottom layer, and wherein the at least one reservoir ofthe bottom layer comprises a test reagent configured to test for atleast one of improvised explosives and homemade explosives.
 18. A methodof testing a sample for explosives, comprising: providing the sample tothe 3-D PMD according to claim
 14. 19. The method according to claim 11,further comprising dissolving the sample in a solvent to give a samplesolution, wherein providing the sample to the 3-D PMD comprisesinjecting the sample solution into a top layer of the 3-D PMD in avolume of less than 75 pt.
 20. A method of manufacturing a papermicrofluidic device, comprising: printing a wax pattern on a papersubstrate, wherein the pattern comprises one or more channels; heatingthe paper substrate so the wax penetrates the paper substrate, such thatthe one or more channels are hydrophobic channels; and providing a testreagent at a test spot of at least one of the hydrophobic channels,wherein the test reagent is configured to test for explosives.